Carcinoembryonic Antigen Expression and Resistance to Radiation and 5-Fluorouracil-Induced Apoptosis and Autophagy.

Understanding the mechanism of tumor resistance is critical for cancer therapy. In this study, we investigated the effect of carcinoembryonic antigen (CEA) overexpression on UV-and 5-fluorouracil (5-FU)-induced apoptosis and autophagy in colorectal cancer cells. We used histone deacetylase (HDAC) inhibitor, NaB and DNA demethylating agent, 5-azacytidine (5-AZA) to induce CEA expression in HT29/219 and SW742 colorectal cancer cell lines. MTT assay was used to measure IC50 value of the cells exposed to graded concentrations of 5- FU with either 0.1 mM NaB or 1 μM 5-AZA for 72 h . Using CHO- and SW742-CEA transfectants, we also investigated the effect of CEA expression on UV- and 5-FU-induced apoptosis and autophagy. Treatment of HT29/219 cell line with NaB and 5-AZA increased CEA expression by 29% and 31%, respectively. Compared with control cells, the IC50 value for 5-FU of NaB and 5-AZA-treated cells increased by 40% and 57%, respectively. Treatment of SW742 cells with NaB or 5-AZA increased neither CEA expression nor the IC50 value for 5-FU. In comparison to parental cells, CEA expression also significantly protected transfected cells against UV-induced apoptosis. Decreased proportions of autophagy and apoptosis were also observed in 5-FU treated SW742- and CHO-CEA transfectants. We conclude that CEA expression can effectively protect colorectal cancer cells against radiation and drug-induced apoptosis and autophagy.

poor tumor response to chemoradiotherapy and an increased risk of relapse (4,5). Previous findings of in vitro and in vivo studies have also suggested that CEA overexpression has an instrumental role in human cancer progression by inhibiting cell differentiation and anoikis, a form of apoptosis caused by detachment from extracellular matrix (6,7). showing that transfection of human CRC cells by CEA cDNA augments resistance to  cytotoxicity in in vitro model (9,10).
While apoptosis is considered as type I cell death program, autophagy is the second important type of physiological cell death which is referred as type II cell death. 5-FU chemotherapy induces both apoptosis and autophagy in colon cancer cells (11)(12)(13). It has been suggested that there is cross-talk between these two types of cell death processes (14,15). Autophagy is believed to play an important role in tumor formation by functioning as a selfdefense mechanism protecting cancer cells against chemotherapy-induced apoptosis (11,16).
In this study, we have investigated the effect of CEA overexpression on resistance to UV and 5-FU-induced autophagy and apoptotic cell death.
Insight in the molecular mechanisms of drug resistance can facilitate the identification of biological markers to predict drug response and therefore improve tumor-selective therapeutic strategy.

Cell lines and transfection
The human colorectal carcinoma cell lines SW742, HT29/219 and the Chinese hamster ovary (CHO) cell line were obtained from the National Cell Bank of Iran (NCBI, Pasteur Institute, Tehran).
All cell lines were cultured in RPMI 1640 supplemented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (all from Gibco) in a humidified 5% CO2 atmosphere at 37 °C. CHO and SW742 cells were stably transfected with full length CEA cDNA using pCDNA3.1 (+) expression vector (Invitrogen, USA), as described previously (9). The CEA protein content of transfected cell lines was determined by commercially available ELISA kit (CanAg Diagnostics AB, Gothenburg, Sweden) and the corresponding band of CEA (180 KD) was shown using western blot.

Cell treatment
HT29/219 and SW742 cells at a density of 2.5 x 10 5 cells were seeded in 6-cm tissue culture dishes under normal culture condition for 24 h. Cells were treated with an increasing concentration of sodium butyrate (NaB) (0, 0.1, 0.2, 0.5 and 1mM) or 5-azacytidine (5-AZA) (0, 0.125, 0.25, 0.5, 1 and 2 µM) for 10 and 72 h, respectively. Cell lysate was prepared as previously described (9) and the CEA content of cells was determined using CEA ELISA kit (CanAg Diagnostics AB, Gothenburg, Sweden). Cell viability was determined by MTT dye reduction assay as described previously (9). The concentration of NaB

Apoptosis analysis
For the apoptosis assay, propideum iodide (PI) staining and DNA fragmentation were used as described previously (17). Briefly, CEA-transfected and control CHO and SW742 parental cells were seeded at a density of 3x10 5 cells in 10 cm tissue culture dishes. After 24 h, CHO and SW742 cells were exposed to 250 µM and 400 µM 5-FU, respectively. After 72 h of 5-FU treatment, cells were trypsinized, and fixed in 70% ethanol for 2 h.

Autophagy detection by acridine orange staining
For autophagy analysis, the development of acidic vesicular organelles (AVO) was quantified as described previously (18). For this purpose, CEAtransfected CHO and SW742 and control parental cells were seeded in 6-cm tissue culture dishes at a density of 3x10 5

Statistical analysis
Data from multiple experiments were expressed as mean ± SD. Differences between groups were tested by Kruskal-Wallis test and Mann-Whitney U test. P< 0.05 was considered to be significant.

CEA protects cells from 5-FU induced apoptosis
Apoptosis has been implicated as one of the mechanism of cell death induced by 5-FU in colon cancer cells (12,16,22 Notably, the extent of DNA fragmentation was greater in control parental cells than CEA transfected cells (Fig. 1). Flow cytometric analysis using propodium iodide staining and flow cytometry was also performed to quantify apoptosis of 5-FU-treated cells. Compared with the control parental cells, CEA transfected CHO and SW742 cells had a significantly lower apoptotic rates (71% and 79% reduced apoptosis, respectively) ( Fig. 2).

Overexpression of CEA protects cells from 5-FU induced autophagy
Authophagy is an alternative type of programmed cell death that is activated in human colon cancer cells after treatment with 5-FU (11,23). Using two CEA-transfected cell lines, SW742 and CHO, we investigated the effect of CEA expression on 5-FU induced autophagy. 5-FU treated cells were stained with acridine orange. As shown in Figure 3A and    The histogram of DNA content distribution of CEA tranfected CHO and SW742 and control parental cells. Cells were treated with 5-FU for 72 h, fixed in ethanol, stained with propidium iodide, and DNA content was determined by flow cytometry. The arrowhead marks the apoptotic peak, namely sub-G1 peak in these cells. CEA transfectants show significantly lower apoptotic rate than their control counterparts.

5-FU
has been the drug of choice for the treatment of CRC patients for several decades.
However, many of CRC patients have tumors intrinsically resistant to 5-FU-induced cytotoxicity.
Previous work in our laboratory has shown that increased stable expression of CEA in cells was associated with increased resistance against 5-FU (expressed as higher IC 50 value) (9). The objectives of the present study were to further investigate how changes in the levels of CEA expression could increase resistance against chemical and physicalinduced cytotoxicity.
It has been reported that HDAC inhibitor, NaB and DNA methyltransferase inhibitor, 5-AZA upregulate CEA expression in different cancer cells (19)(20)(21). Therefore, we examined the effect of NaB Our findings are consistent with previous results, showing that drug-resistant cells from human colorectal adenocarcinoma tumors produce higher than normal levels of CEA per cell (25,26).
Increased expression of CEA in colon carcinoma cells has been shown to be correlated with promoter hypomethylation of this gene in comparison to normal cells (27)(28)(29) (Table 3). There are obviously many other factors other than CEA expression levels that may contribute to the radiation resistance. These include various genetic and epigenetic mechanisms involved in apoptosis, autophagy, and radiationinduced cell injury process (33).
We used DNA ladder assay and flow cytometric analysis of propidium iodide stained cells to assess apoptosis. We found that the relative frequency of apoptosis observed in the parental control cell lines, as verified by DNA laddering assay, was higher than those of CEA-transfected (5-
The relationship between apoptosis and autophagy is also complex. Although, autophagy is an independent mechanism of cell death, there lies upstream of apoptosis and is necessary for the latter to occur (11,16,40).
Although, more sensitive methods such as quantitative measurement of acidic vesicular organelles by flow cytometry and western blot analysis of microtubule-associated protein light chain 3 (LC3) should be used to confirm our results, to our knowledge, this is the first study to demonstrate that CEA expression serves to protect against chemotherapy-induced autophagy.